Sodium 4-[4-[[2-methyl-4-[[(p-tolyl)sulphonyl]oxy]phenyl]azo]anilino]-3-nitrobenzenesulphonate

Genetic toxicity: in vivo

Administrative data

Endpoint:

in vivo mammalian somatic cell study: cytogenicity / bone marrow chromosome aberration

Type of information:

read-across from supporting substance (structural analogue or surrogate)

Adequacy of study:

key study

Study period:

From May 19th to July 02nd, 1982

Reliability:

2 (reliable with restrictions)

Rationale for reliability incl. deficiencies:

study well documented, meets generally accepted scientific principles, acceptable for assessment

Justification for type of information:

The read across approach is detailed into the document attached below.

Data source

Reference

Reference Type:

study report

Title:

Unnamed

Year:

1983

Report date:

1983

Materials and methods

Principles of method if other than guideline:

The substance was tested for clastogenic effects using the mouse micronucleus test, following the method of Salamone et al (1981). Cyclophosphamide was used as a positive control to assess the sensitivity of the test system.

Ref: Salamone M.F., Heddle J.A. and Katz M., 1981. Mutagenic activity of 41 compounds in the in vivo micronucleus assay. Prog Mlutat Res 1 686-697.

GLP compliance:

no

Type of assay:

mammalian germ cell cytogenetic assay

Test material

Test material information

Constituent 1

Reference substance name:

Read Across susbtance

IUPAC Name:

Read Across susbtance

Test animals

Species:

mouse

Strain:

C57BL

Sex:

male/female

Details on test animals or test system and environmental conditions:

TEST ANIMALS
- Source: Animal Breeding Unit, Alderley Park, Macclesfield, Cheshire.
- Age at study initiation: 6 - 8 weeks old.
- Housing: on arrival the mice were housed ten per cage on mobile mouse racks. After acclimatisation the animals were housed five per cage.
- Diet: food (Porton Combined Diet [PCD] supplied by BP Nutrition Ltd), ad libitum.
- Water: ad libitum.

ENVIRONMENTAL CONDITIONS
- Temperature: 22 °C (range 21 - 23 °C).
- Humidity: 58 % (range 39 - 76 %).
- Air changes: 22 air changes per hr.
- Photoperiod: 12 hrs dark / 12 hrs light.

Administration / exposure

Route of administration:

intraperitoneal

Details on exposure:

DOSING FOR MAIN TEST
Animals were dosed with two consecutive intraperitoneal injections, adminstered 24 hours apart, using 1000 and 625 mg/kg as there were no deaths on the LD50 study.

LD50 DETERMINATION
The LD50 was calculated on the deaths observed over a seven day period and was determined on a single daily intraperitoneal dose.
The compound was administered as single intraperitoneal dose at the dose levles of 7000, 750, 500, 250 and 150 mg/kg. 5 animals per dose lavel. The animals were observed daily for deaths.

Frequency of treatment:

Animals were dosed with two consecutive intraperitoneal injections.

Post exposure period:

5 males and 5 females were sacrified after 24 hours from the start of exposure; further 5 males and 5 females after 48 hours; further 5 males and 5 females after 72 hours.

Doses / concentrations

Doses / concentrations 1

Dose / conc.:

1 000 other: mg/kg

Doses / concentrations 2

Dose / conc.:

625 other: mg/kg

Remarks:

The lower dose level was used to ensure a test result in the event of deaths in the top dose group and to allow the observation of a dose response, should the higher dose give a positive result.

No. of animals per sex per dose:

15 males and 15 females

Control animals:

yes

Positive control(s):

Cylophosphamide. Solvent used was physiological saline.
Dose: 60 mg/kg

Examinations

Tissues and cell types examined:

Polychromatic erythrocytes

Details of tissue and slide preparation:

The animals were killed by cervical dislocation at 24, 48 and 72 hours after the last dose of compound.
Femurs were removed and stripped clean of muscle. The iliac end of the femur was removed and a fine paint brush wetted with a solution of albumen (6 % v/v in saline) was dipped into the marrow canal.
3 to 4 streaks of marrow suspension were then applied to appropriately is labelled clean, dry microscope slides. The slides were allowed to air dry.
e The slides were then stained in Wright's stain using an Ames Hema-Tek staining machine (Hema-Tek, Miles Laboratory Limited, Stoke Court, Stoke Poges, Slough, UK).
Slides were coded and scored blind.
500 polychromatic erythrocytes were examined and the number containing micronuclei scored. The samples were also examined for any evidence of cytotoxicity.

Statistics:

The results were analysed for significant differences from the control group using a one sided Student's 't' test.

Results and discussion

Test results

Sex:

male/female

Genotoxicity:

negative

Toxicity:

yes

Remarks:

deaths at the higher dose level

Negative controls validity:

valid

Positive controls validity:

valid

Additional information on results:

Test item gave negative results at all three sampling times, even though there were a number of deaths which occurred in the higher dose level animals. These deaths were unexpected based on the LD50 data and were probably due to administration of the compound as split dose rather than a single treatment. However, the 625 mg/kg dose group would qualify as a maximum tolerated dose at the 48 and 72hr sampling times.

The positive control cyclophoshamide gave an elevated and statistically significant increase in the incidence of micronuclei at both the 24 and 48 hr sampling times.

Any other information on results incl. tables

Incidence of micronuclei/1000 polychromatic erythrocytes at three sampling times

Compound	Dose	Incidence of micronuclei/1000 cells
		24 hrs	48 hrs	72 hrs
Control (corn oil)	0.1 ml/10 g	1.0	2.0	1.9
Cyclophosphamide	60 mg/kg	52.1*	7.6**	2.4
Test item	1000 mg/kg	1.5	1.8	2.0
Test item	625 mg/kg	1.0	2.0	1.3

* Statistically significantly different p < 0.001

** Statistically significantly different p<0.01 (one sided).

Individual animal data

Compound	Dose	Sex	Incidence of micronuclei/1000 cells
			24 hrs	48 hrs	72 hrs
Control (corn oil)	0.1 ml/10 g	Male	0, 2, 2, 0, 1	41, 0, 2, ND, 4	2, 2, 61, 3, 2
		Female	2, 1, 0, 0, 2	3, 4, 0, 2, 2	0, 4, 2, 3, 0
Cyclophosphamide	60 mg/kg	Male	48, ND, 37, 60, 47	10, ND, 8, 1, 6	0, 1, 2, 3, 1
		Female	ND, 65, 47, 63, 50	10, 7, 12, 6, 10	1, 3, 5, 2, 6
Test item	1000 mg/kg	Male	3, 1, 2, 0, ND, 4	3, 1, 2, 0, 4	D, D, D, D, 2
		Female	0, 2, 1, 2, 2	1, 2, D, D, D	D, 2, D, D, D
Test item	625 mg/kg	Male	1, 3, 0, 2, 0	4, 6, 1, 1, 0	0, 0, 3, 2, 0
		Female	1, 0, 2, 0, 1	4, 1, 2, 0, 1	3, 0, 2, 1, 2

ND: sample not counted, insufficient cells or poor sample

D: animal found dead

LD50 DETERMINATION

Compound	Dose (mg/kg)	N. animal	N. deaths
Test item	1000	5	0
Test item	750	5	0
Test item	500	5	0
Test item	250	5	0
Test item	150	5	0

Applicant's summary and conclusion

Conclusions:

The results indicate thatthe substance has no clastogenic activity in the mouse micronucleus test.

Executive summary:

The substance was tested for clastogenic effects using the mouse micronucleus test, following the method of Salamone et al (1981). Cyclophosphamide was used as a positive control to assess the sensitivity of the test system.

The substance was tested for clastogenic activity in the mouse micronucleus test using C57BL/6J mice.

Test item did not induce any statistically significant increase in the frequency of micronuclei when compared with control animals.

Throughout the study the positive control substance, cyclophosphamide, gave the expected responses.

Conclusion

These results indicate that substance is not clastogenic in the mouse micronucleus test.

Ref: Salamone M.F., Heddle J.A. and Katz M., 1981. Mutagenic activity of 41 compounds in the in vivo micronucleus assay. Prog Mlutat Res 1 686-697.